基于iPS的VKH综合征细胞模型的建立、发病机制及药物筛选研究

VKH syndrome is a multiple gene autoimmune disease characterized by bilateral granulomatous uveitis. At present, the understanding of the pathogenesis of this disease is mostly from animal models (EAU), however, the clinical manifestations and species differences of EAU mice and humans limit the application of the results from EAU model in translational researches. The patient's iPS cells carry the whole set of genetic information of VKH patient's somatic cells and meanwhile it can also differentiate into various retinal cells, so hiPSC-derived RPE cells may effectively compensate for these defects. Recently, our group found two new associated genes with VKH syndrome (IL23R and EGR2) in multiple ethnic populations, however, the functional roles and the mechanism of these two genes remain unclear. The current project aims to establish the iPS cell modeling of VKH syndrome through inducing RPE cells by PBMCs from VKH patients and controls and then identify the corresponding phenotype of RPE cells. The membrane resistance of RPE, tight junction protein, phagocytosis roles, protein expression, protein phosphorylation and protein interaction are examined to investigate the functional roles and the mechanism of IL23R and EGR2 in this disease. High-throughput and computer-aided drug screening are used to find the novelty drug for this syndrome, and then EAU is used as in vivo tool to elucidate the functional roles and mechanism of these drugs in the development of VKH syndrome. This study will probably establish the iPS cell-based VKH syndrome modeling, clarify the new mechanism of this disease and find leading compounds of this disease, so as to lay an important foundation for the precise treatment of this syndrome.

VKH综合征是以双侧肉芽肿性全葡萄膜炎为特征的多基因遗传的自身免疫病。目前对该病发病机制的理解多来自动物模型，但动物与人类的临床表现及种属差别限制了其在转化研究中的应用。患者来源iPS细胞携带疾病全套遗传信息同时也能分化成视网膜各类细胞，有效弥补了此项缺憾。团队前期发现了VKH综合征2个新的相关基因-IL23R和EGR2，但其作用机制尚未阐明。基于此，本项目以VKH综合征为研究对象，诱导PBMC经iPS定向分化为RPE并对其进行相应表型鉴定，以期建立该病细胞模型；通过对RPE跨膜电阻、紧密连接蛋白、吞噬功能、蛋白表达、蛋白磷酸化修饰、蛋白互作等研究，探讨IL23R和EGR2在疾病发生中的作用机制；通过高通量和虚拟筛选发现疾病先导化合物，继而通过EAU模型对其进行体内功能及机制验证。本研究将可能建立VKH综合征iPS模型，阐明该病发生的新机制并发现疾病先导化合物，为此病精准防治奠定基础。

我们课题组按照任务书进行系列研究，获得了以下成果：①我们严格按照任务书的内容，在国际上首次构建了人类葡萄膜炎-VKH综合征的细胞模型，以此为基础发现了数种治疗葡萄膜炎的潜在小分子化合物和天然产物单体，为免疫性致盲眼病的防治提供了新思路和新策略。阐明了小胶质细胞功能调控在葡萄膜炎发生中的免疫学机制，发现了由NLRP3、FTO、AhR等“刹车分子”（负调控）和Galectin-3等“油门分子”（正调控）构成的庞大调控网络，为眼免疫病的防控提供了重要分子靶点。在项目资助下，以通讯作者在Arthritis Rheumatol（IF:15.483）、Redox Biol（IF:10.787）、Clin Immunol（IF:10.19，3篇）、Medcomm、Cell Death & Dis、IOVS等发表14篇SCI论文，以第一完成人授权2项国家发明专利。.②培养了2名博士后，3名博士研究生及5名硕士研究生，为免疫性致盲眼病的防控奠定了重要基础。.③强化了与国外著名研究机构的合作，在国内外重要学术会议报告研究结果，进一步扩大了我们在免疫性致盲眼病领域的国际影响力。.④项目执行期间，以项目负责人新获得2项国家自然科学基金面上项目资助；获纪念中华人民共和国成立70周年纪念章；入选重庆市首席专家工作室领衔专家。.综上，本项目共资助发表SCI论文14篇（均为通讯作者），其中IF>10， 5篇。执行阶段，课题负责人获得国家自然科学基金面上项目2项，入选重庆市首席专家工作室领衔专家。

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